Harvey, Kirsten and N ras proteins are immunologically related proteins and are collectively termed p21. They are products of the ras family of cellular genes which are found in a wide variety of nucleated mammalian cells. The ras genes appear to be frequent targets of genetic alterations that can lead normal cells along the pathway to malignancy. Ras oncogenes have been identified in a wide array of premalignant and malignant cells.
The p21 proteins consist of about 188-189 amino acids having a molecular weight of about 21,000 daltons. Viral and cellular ras genes encode membrane bound proteins (Willingham et al., Cell 19;1005 (1980)) which bind guanine nucleotides (Schlonick et al., PNAS (USA) 76:5355 (1979); Papageorge et al., J. Virol. 44:509 (1982); and Fine et al., Cell 37:151 (1984)) and possess intrinsic GTPase activity (McGrath et al., Nature 301:644 (1984); Sweet et al., Nature 311:273 (1984); Gibbs et al., PNAS (USA) 81:5704 (1984); and Manne et al., PNAS 82:376 (1985)).
DNA mediated transfection experiments using NIH3T3 cells as recipients have led to the identification of a family of activated transforming genes homologous to the ras genes of the Harvey (Ha-ras) and Kirsten (Ki-ras) sarcoma viruses. A third member of the ras family designated N-ras has been identified but has not been found to have a retroviral counterpart. Activated (mutated) ras genes are structurally distinct from their normal homologs, having amino acid substitutions in the protein at positions 12, 13, or 61. (Tabin et al., Nature 300:143 (1982); Reddy et al., Nature 300:149 (1982); Bos et al., Nature 315:716 (1985); Yuasa et al., Nature, 303:775-779 (1983); Der et al., Cell 44:167-176 (Jan. 17, 1986)). Taparowsky et al., Banbury Report, 14:123-133 (1983) cited in Chem. Abstracts CA 100(1):1425n, teaches that the change at residue 12 from N-terminus of the H ras p21 from glycine to valine is sufficient to convert the normal protein to a transforming protein.
Shimizu et al., Nature 304:497-500 (1983) cited in Chem. Abstracts 99(19):1530936, teaches the presence of a cysteine residue at amino acid 12 in the human lung cancer cell line calu-1 homolog of the v-Ki-ras gene. Fasano et al., J. Mol. Appl. Genet., 2(2):173-180, cited in Chem. Abstracts CA 99(19):153080v, teaches that the T24 H-ras-1 gene product is nearly identical to the v-H-ras p21 transforming protein encoded by Harvey sarcoma virus. Recent reports have shown the presence of activated ras p21 proteins in 40-50% of human colorectal cancers and preneoplastic lesions of the colon termed adenomas (Bos et al., Nature 327:293 (1987), Forrester et al., Nature 327:299 (1987) and Volgelstein et al., NEJM 319:525 (September 1988)). Recent studies have also shown expression of activated ras genes and mutated ras p21 proteins in 20-30% of lung carcinomas (Rodenhuis et al., Cancer Res., 48:5738 (1988)) and over 90% of pancreatic carcinomas (Almoguera et al., Cell 53:549 (1988)). In certain forms of leukemia such as acute myelogeneous leukemia and in certain preleukemic states, activated ras p21 proteins have been described.
These activated ras genes and mutated proteins have also been found in established cell lines as well as primary and metastatic tumors. Gambke et al., (Nature 307:476, 1984), demonstrated a transforming N-ras gene in bone marrow cells from a patient with acute myeloblastic leukemia (AML). In contrast, DNA from fibroblast cells from the same patient was not transforming.
The p21 ras protein in its normal nonactivated form contains the glycine amino acid at positions 12 and 13 and the glutamine amino acid at position 61. The p21 protein found in normal cells has the following primary amino acid structure for the amino acid sequence 5 to 19: .sup.5 Lysine-leucine-valine-valine-valine-glycine-alanine-glycine-glycine-valine -glycine-lysine-serine-alanine-leucine.sup.19.
Previous reports have described several rat monoclonal antibodies reactive with normal and activated or oncogenic (mutated) ras p21 proteins in yeast and mammalian cells. (Robinson et al., Br. J. Cancer 54:87783-883 (1986), Furth et al., J. Virol. 43:294 (1982)).
EPO Patent Application No. 85111824.0 published on, Apr. 16, 1986, and European Patent Application No. 85111823.2 published on Mar. 26, 1986, disclose a polypeptide consisting of amino acids 5 to 17 of the ras p21 protein containing a cysteine residue inserted between positions 16 and 17 and further containing amino acid substitutions at position 12. Amino acids valine, serine, arginine, cysteine, aspartic acid or alanine were inserted at position 12. These polypeptides were coupled to carrier proteins and used as immunogens to induce the production of antibodies discussed therein. This reference further indicates that antibodies capable of distinguishing ras oncogenes from their normal counterparts by virtue of single amino acid differences in the p21 gene product may be applicable to diagnostic detection of malignant cells in clinical situations and it further indicates that such antibodies capable of distinguishing normal ras p21 from mutant ras p21 having a single amino acid difference at position 12 or 61 "would be used to detect the ras oncogene product by standard techniques such as immunofluorescence, immunoperoxidase staining, immunoprecipitation, ELISA, or Western blotting techniques."
Carney et al., PNAS (USA) 83:7485-7489 (1986) and EPO Publication No. 019003 published on Aug. 6, 1986, disclose a monoclonal antibody specific for an activated ras protein. This monoclonal antibody was raised against a synthetic peptide corresponding to amino acids of a mutated ras gene encoding valine instead of glycine at position 12. EPO Publication No. 019003 mentions that monoclonal antibody DWP is useful in the diagnosis of primary and metastatic lesions by conventional diagnostic methods and that diagnosis can also be carried out by conventional in vitro diagnostic procedures such as the assay of human blood samples or other bodily fluids. Carney et al., UCLA Symp. Mol. Cell. Biol., New Ser. 1985 cited in Chem. Abstracts, CA 104:1665706, disclose a monoclonal antibody raised against a ras related synthetic peptide showing immunoreactivity with human carcinomas. Carney et al. reported a series of monoclonal antibodies raised against synthetic peptides containing amino acid substitutions of glutamic acid, arginine or valine at position 12 (A Book of Abstracts from the 3d Annual Meeting on Oncogenes held at Hood College, Frederick, M.D., Jul. 7-11, 1987). Other monoclonal antibodies generated by various methods have also been reported to react with the various forms of the ras p21 protein. Hand et al., Proc. Nat. Acad. Sci. USA, Vol. 81, pp. 5227-5231 (1984); Thor et al., Nature, Vol. 311, pp. 562-565 (1984); Wong et al., Cancer Research, Vol. 46, pp. 6029-6033 (1986); and Tanaka, Proc. Natl. Acad. Sci. USA, Vol. 82, pp. 3400-3404 (1985).
Several scientific reports have shown that normal cells contain ras proteins with glycine at position 13.
In 1985 Bos et al. (Nature 315:726 1985) demonstrated that DNA isolated from cells of AML patients were able to transform NIH3T3 cells. This result is indicative and highly suggestive for the presence of an oncogene. These transforming genes were shown to be activated ras genes. In contrast, DNA from normal tissues were non-transforming and therefore did not contain activated N ras. These investigators analyzed the activated N ras genes for the presence of mutations using oligonucleotide probes and found that the activated N ras genes contain mutations that result in amino acid substitutions at position 13 of the protein. These mutations at position 13 were shown to be either aspartic acid or valine instead of the normal amino acid glycine.
Two reports in 1987 described ras mutations with arginine at position 13. Nitta et al. have shown (Jpn. J. Cancer Res. (Gann), 78:21-26 1987) an amino acid substitution of arginine for glycine at position 13 of an activated N ras p21 isolated from a human rectal carcinoma. A report by Hirai et al. (Nature 327:430 1987) has shown activated N ras genes in bone marrow cells from patients with myelodysplastic syndrome. The observations made by Hirai et al. suggest that the presence of activated N ras genes with position 13 mutations may be important in early stages of leukemia.
A report by E. Liu et al. (Nature 330:186, 1987) demonstrated the presence of the aspartic acid mutation at position 13 of the ras p21 in a patient with myelodysplastic disease 1.5 years before the patient progressed to acute leukemia. Thus screening patients with myelodysplastic syndrome for the presence of activated ras proteins with position 13 mutations with monoclonal antibodies may be a valuable test to predict which patients with myelodysplastic syndrome have an increased risk of developing acute leukemia.
Most recently, Wodnar-Filipowicz et al. reported (Oncogene, pp. 457-461, Vol. 1, No. 4 (1987)) the presence of activated N ras genes in a human T cell non-Hodgkin's lymphoma. These studies demonstrated the substitution of cysteine for glycine at position 13.
Reports have suggested that the 188-189 amino acid sequence of the H, Ki, and N ras p21s have been greatly conserved throughout evolution. However, the most significant differences between the H, Ki, and N p21 proteins appear to be localized in a segment having 15-20 amino acids located at the carboxy end of the p21 protein (Taparowsky et al., Nature 300:762 (1982)).
Furthermore, McGrath et al., Nature 304:501-506 (1983), and Shimizu et al., Nature 304:497-500 (1983), have shown that the Ki gene has the capacity to encode two distinct proteins referred to as Ki4A and Ki4B. The terms Ki4A and Ki4B are used interchangeably with Ki2A and Ki2B. Since this variable region exists among amino acids 160-180 at the C-terminal end of the ras p21, then it is theoretically possible to generate monoclonal antibodies that could specifically bind the individual H, Ki4A, Ki4B, and N ras p21s. Since reports indicate that activation of a particular ras gene such as the N ras gene in acute myelogenous leukemia (Bos et al., Nature 315:726 (1985)) or the overexpression of the particular H-ras gene (Spandidos et al., Anticancer Res. 4:269 (1984)) is frequently associated with a specific type of cancer (breast cancer).
European Patent Application No. 86107244.5, published on Dec. 3, 1986, discloses polypeptides having amino acid sequences derived from the variable regions of ras.sup.H, ras.sup.N, ras.sup.4KA and ras.sup.4KB protein families, immunogenic compositions wherein these polypeptides are covalently attached to immunogenic carriers, antibodies produced from such immunogens wherein these antibodies are specific for the ras oncogene from which the polypeptide sequence was derived and immunoassays employing these antibodies to distinguish among the individual p21 ras oncogene families. The peptide structures disclosed correspond to amino acids 171-189 and 170-189 of p21 H ras, 170-186 of p21 N ras, 171-186 of p21 Ki4A ras, and 170-185 of p21 Ki4B ras. It appears that no antibodies were deposited.
U.S. Pat. 4,535,058, issued Aug. 13, 1985 to Weinberg et al., discloses the general concept of using hybridoma technology to produce monoclonal antibodies to altered forms of ras p21 proteins or peptides encompassing position 12 of the proteins. In particular, attention is invited to column 4, lines 6-15, column 12, line 33 through column 13, line 29, and column 14, line 40 through column 16, line 22.
U.S. Pat. 4,699,877, issued Oct. 13, 1987 to Cline et al., discloses methods and compositions for detecting human tumors. A series of oligopeptides corresponding to antigenic regions in the peptide expression products of RNA present in retrovirus oncogenes is disclosed in column 5 at lines 17-60. Included in this series are Ras.sup.Ki and Ras.sup.Ha oligopeptides. This reference further discloses that these haptenic oligopeptides be used to induce antibody formation by coupling to an appropriate carrier. The method disclosed looks to cellular products such as mRNA or its expression product as diagnostic of the probable presence of malignant cells.
Tanaka et al., PNAS 82:3400 (1985), reported the generation of a series of rabbit sera to a variety of synthetic peptides corresponding to various portions of the ras p21s. Tanaka et al. reported the production of rabbit sera to a peptide corresponding to amino acids 160-179 of the v-Ha-ras. The anti-p21 sera was prepared by affinity purifying the rabbit sera and evaluating their specificity by biochemical assays. The specificity of these reagents, however, is questionable.
Srivastava et al., Molecular and Cellular Biology 5(11):3316 (1985), reported a series of rabbit polyclonal sera to synthetic peptides corresponding to various segments of the protein and in particular a segment corresponding to amino acid 161-176 of the H ras p21.
Tahara et al., Jpn. J. Cancer Res. (Gann) 77:517-522 (1986), disclose that a sheep anti-p21 antibody was generated against a synthetic peptide corresponding to positions 160-179 of the v-Ha-p21.
Bizub et al., Oncogene 1:131-142 (1987), raised antisera in mice, rats and rabbits to a variety of peptides in the H, Ki and N ras variable regions. Some of the polyclonal antibodies described in this report were affinity purified rabbit sera raised against peptides corresponding to amino acids 171-189 of the H p21 or to peptides corresponding to amino acids 171-186 of the Ki4B p21. According to this report, additional antibodies to the ras p21s are available at the N.C.I. repository (Microbiological Assoc. Inc., Bethesda, Md.). The antisera mentioned in the Bizub et al. report indicated that antibodies available at the N.C.I. were raised against a peptide structure correlating to the amino acid sequence 157-180 of the ras p21s.
Hand et al., JNCI, 79(1):59-65 (July 1987) disclose direct-binding liquid competition assays using monoclonal antibody Y13-259 and immunohistochemical assays in concert with cDNA probes for identification of specific ras point-mutated oncogenes or proto-oncogenes may be a possible means to quantitate ras p21 in human carcinomas and benign lesions. Monoclonal antibody Y13-259 discussed in Furth et al., J. Virol. 43:294-304 (1982) is a rat monoclonal generated against the native form of v-Ha-ras p21.
Similarly, Ohuchi et al., Cancer Research 47:1413-1420 (Mar. 1, 1987) disclose enhanced expression of c-Ha-ras p21 in human stomach adenocarcinomas defined by immunoassays using monoclonal antibodies and in situ hybridization. Specificity of the monoclonal antibodies used is questionable.
Caruso et al., Int. J. Cancer 38:587-595 (1986), disclose quantitative analysis of ras p21 in mammalian cells using monoclonal antibodies Y13-259 as discussed above and Y13-238, a rat monoclonal which selectively immunoprecipitate the Ha-MuSV-encoded ras p21.
Niman et al., PNAS (USA) 82:7924-7928 (1985), disclose the use of anti-peptide antibodies to detect oncogene-related proteins in urine. Increased levels of oncogene-related proteins were found during neoplasia and pregnancy. The peptide fragments were selected because they represented highly conserved regions of their oncogene families--sis, ras, and fes. The ras peptide was the Ha ras sequence located 37-59 amino acids downstream from the threonine residue autophosphorylated by p21 encoded by v-Ha-ras or v-Ki-ras. Detection of a 21,000 dalton protein was reported. However, it is not clear whether this protein was a ras related protein due to the questionable specificity of the reagents used.
PCT Application having International Publication Number WO 85/00807, published on Feb. 28, 1985, describes the production of polypeptide-induced monoclonal antibodies to oncoproteins and their use in diagnostic systems to assay for the presence of an oncoprotein.